The Preliminary Study On Molecular Mechanism Of Salt Tolerance In Hibiscus Hamabo Sieb.et Zucc

Hibiscus hamabo Sieb.et Zucc,a kind of deciduous small tree,belonging to Malvaceae Hibiscus,has strong tolerance to salinity and can grow in alkaline(pH8.0 ~ 8.6)soil in which the content of salt is up to 1.5 %.At present,the researches of the Hibiscus hamabo Sieb.et Zucc salt resistance are mainly focused on physiological point of view,but few studies on molecular mechanism.In this study,we analyzed the molecular mechanism of salt tolerance of Hibiscus hamabo Sieb.et Zucc,and wanted to understand the molecular regulation mechanism of salt tolerance of Hibiscus hamabo Sieb.et Zucc.We hoped to promote the comprehensive utilization of Hibiscus hamabo Sieb.et Zucc.1.Analysis and validation of transcriptome sequencing of Hibiscus hamabo Sieb.et Zucc unde r salt stressHibiscus hamabo which had 10 true leaves was subjected to salinity(200 mM NaCl)for 1d,the untreated was kept as control,and then took the leaves and roots to transcriptom analysis.The transcriptome sequencing of four samples was completed.A total of 20.52 gigabases(Gb)clean date were generated from the sample c DNA libraries with 93% at the Q30 level.After de novo assembly we obtaining a total of 75,257 unigenes,and there were 17,795 unigenes with length >1kb.The obtained unigenes were compared with NR,Swiss-Prot,K EGG,COG,KOG,GO and Pfam databases,and 44,483 results were obtained.We gained 6,271 SSR markers based on the gene structure analysis of unigene database.We did the genes mode clustering analysis,functional annotation and enrichment.1,268 differentially expressed genes(DEGs)of the leaves were tested out by RNA sequencing(RNA-seq)treatment with 200 mM NaCl for 1d.Among these DEGs,375 genes were up-regulated and 893 genes were down-regulated.2,093 DEGs of the roots were tested.Among these DEGs,935 genes were up-regulated and 1,104 genes were down-regulated.Simultaneously,seventeen genes were selected for quantitative PCR and the reliability of the transcriptome data were demonstrated,indicating that the data of the transcriptome can be used for further study.2.The analysis of DEGs expression and the measurement of some physiological characteristics of Hibiscus hamabo which was under salt stressWe seclected four DEGs(APX,GST,SOD and AKT1)that related to saltresisitant according to the RNA sequencing(RNA-seq),and then measured their expression.From the results,we found that these four DEGs expression of Hibiscus hamabo leaves rised in the first stage,and then decreased.The largest expression was at the third day.In the roots,the tend of APX,GST,SOD,and AKT1 expression basically appeard rise,fall,and then rise.We measured APX,SOD,GST enzymatic after salt treated for 0 d,1 d,3 d,6 d,9 d.Found that in the leaves the activity of APX and SOD appeard rise and then fall.The activity of GST showed a rising-down-rising trend.APX activity reached maximum at the sixth day.GST,SOD activity reach maximum at the third day.However in the roots the difference of the APX enzymatic was not remarkable.GST enzymatic in general showd a downward trend.SOD enzymatic apprear rise and then fall,reach the maximum at the sixth day.Above all,when plants was under stress,the genes was expressed at first,then through transcription and translstion generated related protein to resist the stress environment.200 mM NaCl treated for 0 d,1 d,3 d,6 d,9 d,we measured Na+,K+ content,found that in the leaves the content of Na+ gradually rised with time,the content of K+ rised in the first stage,and then decreased.In the roots Na+ content gradually rised with time,K+content appeared gradually fall.3.Proteomic analysis of Hibiscus hamabo Sieb.et Zucc unde r salt stressThe Hibiscus hamabo Sieb.et Zucc was treated with 200 mM NaCl,and one day later,leaves were collected from the same leaf position of the treated and control plants.The leaves was used for protein extraction.The protein samples were separated by 2-DE(two-dimensional electrophoresis),and three biological replications were performed.Then we used PDQuest to analyzed the gel.After exposed to 200 mM NaCl for 1 d,86 protein spots showed significant differences,including the increase in protein abundance of 54,the decrease in protein abundance of 32.The next step,we did the mass spectrometry analysis.77 kinds of differentially expressed proteins(DEPs)were identified.There were 48 kinds of DEPs upregulated and 32 kinds of DEPs down-regulated.The identified proteins coverd a wide range of molecular functions,including photosynthesis,metabolism,protein folding,protein synthesis,processing and degradation,energy,signal,oxidation resistance,membrane intracellular transport,cellular structure.These findings indicated that the DEPs played important roles in resisting salt stress in Hibiscus hamabo Sieb.et Zucc.